Fig 4. Changes in caspase 3 and phosphatidylserine (PS) transposition after rosiglitazone supplementation of the thawing media.

Commercial frozen doses of stallion sperm were thawed and processed as described in the Material and Methods. Split samples were incubated in the presence of rosiglitazone 0 and 75 μM and caspase 3 activity was determined by flow cytometry. Data represent percent changes with respect the controls after 1 hour (A) and two hours (B) of incubation and are expressed as the means ± SEM, observed (* P<0.05, results are derived from three independent frozen ejaculates from 6 different stallions n = 18). F) Western blot (WB) controls for caspase 3 using frozen and thawed stallion spermatozoa as positive controls; semen was processed and analyzed as described in reference 50. (G) Further controls were obtained after incubating stallion spermatozoa at 37°C for 3 hours in the presence of three known inductors of apoptosis, staurosporine 10 μM, thapsigargin 50 μM and betulinic acid 50 μM. In C and D, representative cytograms of the simultaneous detection of active caspase 3 and PS transposition are presented where Q2 and Q3 represent events positive both for caspase 3 and Annexin-V. Q2 represents events with higher caspase 3 expression. No significant changes were detected. In E a heat map showing the intensity of Annexin-V staining demonstrates that PS is preferentially expressed in caspase 3 positive cells.